1. Field of the Invention
This invention relates generally to mammalian stem cells and to differentiated or partially differentiated cells derived therefrom using amphiphilic lipid compounds, and preferably, using novel ceramide analogs of the β-hydroxyalkylamine type. The present invention also relates to methods of producing, differentiating and culturing the cells of the invention, and to uses thereof. The methods alternatively comprise modulating the levels of apoptotic regulating factors, such as PAR-4, and/or modulating the intracellular concentration of endogenous lipid second messengers, such as ceramide. These further methods can optionally be performed in the presence of amphiphilic lipid compounds, and optionally employ MEDII conditioned medium. The invention further relates to compositions comprising the amphiphilic lipid analogs and MEDII conditioned medium.
2. Background Art
Embryonic stem (ES) cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation of mammals and resultant commercial, medical and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells suited to transplantation for treatment of diseases that result from cell damage or dysfunction. Other pluripotent cells and cell lines including early primitive ectoderm-like (EPL) cells as described in International Patent Application WO 99/53021, in vivo or in vitro derived ICM/epiblast, in vivo or in vitro derived primitive ectoderm, primordial germ cells (EG cells), teratocarcinoma cells (EC cells), and pluripotent cells derived by dedifferentiation, reprogramming or by nuclear transfer will share some or all of these properties and applications.
Human ES cells have been described in International Patent Application WO 96/23362, and in U.S. Pat. Nos. 5,843,780, and 6,200,806; and human EG cells have been described in International Patent Application WO 98/43679, and U.S. Pat. No. 6,245,566.
The ability to tightly control differentiation or form homogeneous populations of partially differentiated or terminally differentiated cells by differentiation in vitro of pluripotent cells has proved problematic. Most current approaches involve the formation of embryoid bodies from pluripotent cells in a manner that is not controlled and does not result in homogeneous populations. Mixed cell populations such as those in embryoid bodies of this type are generally unlikely to be suitable for therapeutic or commercial use.
Uncontrolled differentiation produces mixtures of pluripotent stem cells and partially differentiated stem/progenitor cells corresponding to various cell lineages. When these ES-derived cell mixtures are grafted into a recipient tissue the contaminating pluripotent stem cells proliferate and differentiate to form tumors, while the partially differentiated stem and progenitor cells can further differentiate to form a mixture of inappropriate and undesired cell types. It is well known from studies in animal models that tumors originating from contaminating pluripotent cells can cause catastrophic tissue damage and death. In addition, pluripotent cells contaminating a cell transplant can generate various inappropriate stem cell, progenitor cell and differentiated cell types in the donor without forming a tumor. These contaminating cell types can lead to the formation of inappropriate tissues within a cell transplant. These outcomes cannot be tolerated for clinical applications in humans. Therefore, uncontrolled ES cell differentiation makes the clinical use of ES-derived cells in human cell therapies impossible.
Selection procedures have been used to obtain cell populations enriched in neural cells from embryoid bodies. These include genetic modification of ES cells to allow selection of neural cells by antibiotic resistance (Li et al., 1998 Current Biol., 8:971-974), and manipulation of culture conditions to select for neural cells (Okabe et al., 1996 Mech., Dev. 59:89-102; and Tropepe et al., 2001 Neuron, 30:65-78; O'Shea, 2002 Meth in Mol., Biol. 198:3-14). Previously, one research group has demonstrated efficient differentiation of mouse and primate ES cells to TH+ neurons following co-culture with the PA6 stromal cell line, but this technique is not likely to be useful for cell therapy applications as it introduces xenograft issues associated with exposure to non-human cell lines and removal of potential PA6 cell contamination in subsequent cultures (Kawasaki et al., 2000 Neuron, 28:31-40; Kawasaki et al., 2002 Proc. Natl. Acad. Sci. USA, 99(3):1580-1585). Furthermore, the PA6 differentiation procedure generated non-neural terminally differentiated cell types, such as retinal epithelial cells, reducing the usefulness of the cell cultures for cell therapy. In addition, McKay has demonstrated efficient differentiation of mouse ES cells to TH+ neurons, but this differentiation required over-expression of the Nurr-1 transcription factor in combination with exposure to Sonic Hedgehog and FGF8 (Kim et al., 2002 Nature 418(6893):50-6). Furthermore, the McKay protocol involves a complex, five stage differentiation method for differentiation of mouse ES cells to neurons.
In all of these procedures, the differentiation of pluripotent cells in vitro does not involve biological molecules that direct differentiation in a controlled manner. Similarly, in experiments examining neural differentiation from human ES cells, there is no way to control the neural differentiation, and the methods merely allow for the passive development of neural cell types (see Zhang et al., 2001 Nature Biotech, 19(12):1129-1133, and Reubinoff et al., 2001 Nature Biotech, 19(12):1134-40). Hence homogeneous, synchronous populations of neural cells with unrestricted neural differentiation capability are not produced, restricting the ability to derive essentially homogeneous populations of partially differentiated or differentiated neural cells. Another research group differentiated human ES cell derived embryoid bodies in 20% serum containing medium for 4 days followed by plating and selection/expansion of neural cell types in medium containing B27 and N2 supplements (serum free), EGF, FGF-2, PDGF-AA, and IGF-1 (Carpenter et al., 2001 Exper. Neuro., 172:383-397). Carpenter et al. showed that neural progenitors could be enriched from this culture system by cell sorting or immunopanning using antibodies directed against polysialated NCAM or the cell surface molecule recognized by the A2B5 monoclonal antibody.
Chemical inducers such as retinoic acid have also been used to form neural lineages from a variety of pluripotent cells including ES cells (Bain et al., 1995 Dev. Biol., 168:342-357, Strubing et al., 1995 Mech. Dev., 53:275-287, Fraichard et al., 1995 J. Cell Sci., 108:3181-3188, Schuldhrer et al., 2001 Brain Res., 913:201-205; Esdar et al., 2001 Eur. J. Cell Biol., 80:539-553). However, the route of retinoic acid-induced neural differentiation has not been well characterized, and the repertoire of neural cell types produced appears to be generally restricted to ventral somatic motor, branchiomotor or visceromotor neurons (Renoncourt et al., 1998 Mech. Dev., 79:185-197).
Previous publications report the transplantation of ES-derived neural cells into the ventricles of the fetal or newborn rat or mouse brain without the formation of tumors (Brustle et al., 1997 PNAS, 94:14809-14814, Zhang et al., 2001 Nature Biotech, 19:1129-1133). Although some of the cells in these studies do integrate into the host brain, many of the cells in the transplants form neural tube like structures within the lumen of the brain ventricle. Therefore, these previous studies do not lead to methods that can be readily applied to human cell therapy. Note that Reubinoff et al. (2001 Nature Biotech, 19:1134) also injected ES-derived neural cells into the ventricles of newborn mice but did not report intraventricular masses of neural cells, omitting any mention of the presence or absence of such masses.
Neural stem cells and precursor cells have been derived from fetal brain and adult primary central nervous system tissue in a number of species, including rodent and human (e.g., see U.S. Pat. No. 5,753,506 (Johe), U.S. Pat. No. 5,766,948 (Gage), U.S. Pat. No. 5,589,376 (Anderson and Stemple), U.S. Pat. No. 5,851,832 (Weiss et al.), U.S. Pat. No. 5,958,767 (Snyder et al.) and U.S. Pat. No. 5,968,829 (Carpenter). However, each of these disclosures fails to describe a predominantly homogeneous population of neural stem cells able to differentiate into all neural cell types of the central and peripheral nervous systems, and/or essentially homogeneous populations of partially differentiated or terminally differentiated neural cells derived from neural stem cells by controlled differentiation. Furthermore, it is not clear whether cells derived from primary fetal or adult tissue can be expanded sufficiently to meet potential cell and gene therapy demands. Neural stem cells derived from fetal or adult brain are established and expanded after the cells have committed to the neural lineage and in some cases after the cells have committed to neural sublineages. Therefore, these cells do not provide the opportunity to manipulate the early differentiation processes that occur prior to neural commitment. Pluripotent stem cells provide access to these earliest stages of mammalian cellular differentiation opening additional options for cell expansion and directed development of the cells into desired lineages.
It has been suggested that sphingosine, ceramide and ceramide analogs can be used to induce apoptosis in certain cells; however, the results to date have been inconsistent. Ceramide has been reported to induce apoptosis in some cells or cell-lines of neural origin, while in other reports ceramide application has protected the cells from apoptosis. For example, compare Marcora et al., 1996 Found. Clin. Immunol., 4:11-13; Hartfield et al., 1997 FEBS Lett, 401:148-152; Casaccia-Bonnefil et al., 1996 Nature, 383:716-719; Brugg et al., 1996 J. Neurochem., 66:733-739 to Furuya et al., 1998 J. Neurochem., 71:366-377; Irie and Hirabayashi, 1998 J. Neurosci. Res., 54:475-485; Ito & Horigome, 1995 J. Neruochem., 65:463-466; and Liu et al., 2000 Am. J. Cell Physiol., 278:C144-153. The results achieved have been dependent on cell type, cell density, and the concentration of ceramide or the ceramide analog used (For review, see Toman et al., 2002 J. Neurosci. Res., 68:323-330). These experiments have been performed on tumor cells, immortalized cell lines, and primary cultures of differentiated cells, and the results have not been extrapolated to a culture of undifferentiated stem cells. See, Obeid et al., 1993 Science, 259:1769-1771; Marcora et al., 1996 Found. Clin. Immunol., 4:11-13; Hartfield et al., 1997 FEBS Lett, 401:148-152; Casaccia-Bonnefil et al., 1996 Nature, 383:716-719; Herget et al., 2000 J. Biol. Chem., 275:30344-30354; and U.S. Pat. No. 6,410,597 (Bieberich). It has been postulated that the effects of ceramide application may be mediated by a ceramide-activated pathway or feedback mechanism (Jaffrezou et al., 1998 FASEB J., 12:999-1006.)
The complexity of ceramide-dependent neuronal apoptosis, and the resulting lack of predictability in the prior art is demonstrated by a study showing the expression profile of 239 genes that respond to C2-ceramide treatment (Decraene et al., 2002 Genome Biol., 3(8):research 0042.1-0042.22). This study showed that C2-ceramide treatment both upregulated and downregulated pro-apoptotic genes in neuronally differentiated PC12 cells, indicating a complex and unpredictable genetic response to C2-ceramide treatment. Further, certain ceramide experiments have even shown that ceramide induces apoptosis in some differentiated neural cells (See Toman et al., 2002 J. Neurosci. Res., 68:323-330; Brugg et al., 1996 J. Neurochem., 66:733-739), suggesting that the use of ceramide may not be a suitable way to select for differentiated or partially differentiated neural cells that can differentiate into all neural cell types of the central and peripheral nervous systems.
Extensive programmed cell death/apoptosis occurs during neural differentiation in the developing mammalian central nervous system (Chun, J., 2000 Trends in Neuroscience, 23:407-408; Blaschke et al., 1996 Development, 122:1165-1174; Blaschke et al., 1998 J. Comparative Neurology, 396:39-50). This early cell death appears to be largely confined to the neural stem cell and neural progenitor cell pools. In embryonic day 14 mouse cerebral cortex, 70% of the cells undergo apoptosis/programmed cell death (Blaschke et al., 1996 Development, 122:1165-1174). The finding that a large proportion of neural progenitor cells undergo programmed cell death suggests that neural stem/progenitor cells may be extremely responsive to inducers of apoptosis/programmed cell death such as ceramide and ceraride analogs In this context, the resistance of embryonic stem cell-derived neural progenitor/stem cells to ceramide induced or enhanced cell death would be surprising and unexpected.
In summary, it has not been possible to control the differentiation of pluripotent cells in vitro, to provide homogeneous, synchronous populations of neural cells with unrestricted neural differentiation capacity. Similarly, methods have not been developed for the derivation of neural cells from pluripotent cells in a manner that parallels their formation during embryogenesis. In addition, current methods have relied upon the expression of foreign genes to drive neural differentiation of pluripotent stem cells (Kim et al., 2002 Nature, 418:50-56). These limitations have restricted the ability to form essentially homogeneous, synchronous populations of partially differentiated and terminally differentiated neural cells in vitro, and have restricted their further development for therapeutic and commercial applications.
There is a need, therefore, to identify methods and compositions for the production of a population of cells enriched in neural stem cells and the products of their further differentiation, and in particular, human neural cells and their products.